This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To advance human embryonic stem cells and their derivatives towards clinical application for treatment of blood disease. Understanding the mechanisms that regulate hematopoietic stem cell development is essential for further improvement of hematopoietic stem cell use in oncology and gene therapy. The overall goal of this project is to identify and characterize the earliest hematopoietic progenitors in humans to specify cellular and molecular pathways leading to hematopoietic stem cell development using in vitro hESC differentiation system as a model. Directed hematopoietic differentiation of ES cells reproduces many aspects of embryonic hematopoiesis, and provides a unique opportunity to study molecular and cellular pathways of hematopoietic development in humans. In our laboratory, we established a system for efficient hematopoietic differentiation of hES cells through coculture with OP9 bone marrow stromal cells. Using this system we were able to directly differentiate hES cells into cells of all major blood lineages (erythroid, myeloid and lymphoid), as well as identify different stages of hematopoietic commitment. We found that the earliest hematopoietic progenitors (HPs) in humans arise within CD34+ population and could be ultimately defined by surface expression of leukosialin (CD43). In addition, within CD43+ population, we identified lin-CD34+CD43+CD45- hematopoietic progenitors capable of differentiating toward all blood lineages including lymphoid cells, suggesting their hierarchical proximity to hematopoietic stem cells. However, molecular profiling of hES lin-CD34+CD43+CD45- cells revealed altered expression of genes associated with hematopoietic stem cell self-renewal and survival, reflecting limited engraftment potential of ES cell-derived hematopoietic progenitors. With increasing interest in potential therapeutic application of hES cell derivates, identification of genes essential for hematopoietic stem cell development and diversification is of particular importance. The described experimental system sets a solid platform to advance in this direction. This Research used WNPRC stem cell resources and federally approved hES cell lines.